1. Field of the Invention
The present invention relates to a capillary electrophoretic apparatus comprising a capillary electrophoretic part electrophoresing a sample injected into an end of a capillary column and detection means detecting each component separated in the capillary column at an appropriate position of the capillary column, and inclusively, it relates to a method and an apparatus characterized in sample injection into a capillary column.
2. Description of the Prior Art
Besides having those that comprise a single capillary column, capillary electrophoretic apparatuses also have a multi-capillary electrophoretic apparatus comprising a multi-capillary array migration part including a plurality of capillary columns for injecting a plurality of samples one by one into the capillary columns and simultaneously electrophoresing the samples in all capillary columns and an optical measuring part irradiating the capillary columns with light in the multi-capillary array migration part for measuring absorbance by the samples on the irradiated portion or fluorescence from the samples.
The capillary columns include those charged and not charged with gels for migration.
A capillary electrophoretic apparatus is applied for the separation of protein, and sequence determination for DNA. When applied to sequence determination for DNA, the capillary electrophoretic apparatus employs Sanger""s reaction, electrophoreses a DNA fragment sample prepared by labeling a primer or a terminator with a fluorescent material and detects fluorescence from the DNA fragment sample during migration for determining the base sequence.
A DNA sequencer having high sensitivity, a high speed and high throughput is necessary for sequence determination for DNA having long base sequence such as a human genome. As one method, a multi-capillary DNA sequencer prepared by arranging a plurality of capillary columns in place of that employing a flat plate type slab gel is proposed. With such a capillary column, a sample can not only be readily handled or injected but also migrated at a high speed and detected in high sensitivity, when compared to the slab gel. If a high voltage is applied to the slab gel, a band is spread or a temperature gradient is caused due to influence by Joulean heat However, the capillary column hardly causes such problems and can perform detection in high sensitivity with small band spreading even if performing high-speed migration with application of a high voltage.
In capillary electrophoresis, a sample is injected into a capillary column under pressure or with application of a voltage. A method of electrophoretically injecting a sample is widely employed in general due to the simplicity of the apparatus structure, easiness of operations and excellent controllability of parameters.
In relation to the method of injecting the sample into the capillary column, migrational separability in later migrational separation must not be deteriorated and sample injection must be simpler.
Whether the capillary column is charged with a gel or not, one end of the capillary column must be dipped in a prepared sample, the other end must be dipped in a buffer solution, and an electrode such as a platinum wire must be dipped in the vicinity of the end of the capillary column within the sample. In a structure holding the electrode in the vicinity of the end of each capillary column in electrophoretic sample injection, the electrode structure for sample injection becomes complicated in the case of a multi-capillary electrophoretic apparatus collectively arranging a plurality of capillary columns in the form of an array for simultaneously performing electrophoresis. After dipping the end of the capillary column in a sample contained in a sample injection container and performing injection by applying a voltage or the like, the end of the capillary column must be transferred into a reservoir containing a buffer solution for migration. Thus, operations between sample injection and the start of migration are troublesome, and it is convenient if the operations can be automated.
In both of an electrophoretic apparatus comprising a single capillary column and a multi-capillary electrophoretic apparatus, migrational separability may be deteriorated in capillary electrophoresis charged with a gel, depending on the sample injection method. In the case of electrophoretically injecting a sample, a prescribed high voltage is applied within several seconds when starting voltage application and the high voltage is zeroed within several seconds when finishing voltage application in a conventional voltage application method, as shown in FIG. 4. Referring to FIG. 4, the vertical axis shows the voltage (kV) and the horizontal axis shows the time (second). In this example, a voltage of 7.6 kV is applied for 30 seconds. However, If a high voltage is applied to the capillary column, remarkable stress is applied to a gel located on an end of the capillary column due to heat generation or the like to hinder injection of the sample into the capillary column, leading to such bad influence that the number of bases separable in migrational separation is limited. Furthermore, if the high voltage is abruptly applied, the gel may be forced out from the end of the capillary column due to electroosmosis flow. If the forced-out gel is damaged, the migrational separation state is disadvantageously deteriorated.
In slab gel electrophoresis, a large molecule (referred to as a macromolecule) such as template DNA having a size unreceivable in a gel matrix does not enter the gel matrix part but remains on an inlet for the gel matrix part Even if force moving the macromolecule by driving force of an electric field is applied to a separation part in slab gel electrophoresis, the separation part has sufficient resistance against this force due to its sufficient volume to hardly become a problem. Also, in capillary electrophoresis, a macromolecule does not enter a gel matrix part but remains on an inlet for the gel matrix part (this phenomenon is referred to as clogging). Capillary electrophoresis has the following features: 1) the volume of a separation carrier is extremely small, 2) the material for the separation carrier is not a strong one such as an acrylamide gel but a viscous polymer solution, and 3) electric field strength per unit length is large. Therefore, force moving the macromolecule by driving force of an electric field is large, and resistance against this force is small. Thus, occurrence of clogging results in deterioration of a separation pattern.
In DNA sequence determination, since a macromolecule in a sample is template DNA, in order to solve the aforementioned problem resulting from clogging, therefore, the template DNA must be removed before injecting the sample into a capillary column. It is possible to remove the template DNA by chemical pretreatment For example, when preparing a DNA fragment sample by the Sanger""s method, an enzyme, antigen or antibody is bonded to a primer for separating the DNA fragment sample from a template by enzyme reaction or antigen-antibody reaction after preparing the sample. However, such chemical pretreatment is troublesome and requires much labor and time.
The first objective of the present invention is to provide a multi-capillary electrophoretic apparatus simplifying sample injection.
The second objective of the present invention is to provide, in relation to a capillary electrophoretic apparatus, a sample injection method and an apparatus suppressing deterioration of migrational separability.
In order to attain the first objective of simplifying sample injection of a multi-capillary electrophoretic apparatus, a specific sample plate is employed in the present invention. The sample plate comprises a disposable insulating resin base plate including one or a plurality of holes formed as wells having open bottom portions projecting flush with each other, and an electrode plate of conductive metal including cavities having smaller sizes than the wells formed on positions corresponding to two-dimensional arrangements of the wells formed by arrangement of the base plate for receiving press-fitted bottom portions of the wells thereby fixing the base plate. The wells of the base plate are press-fitted in and fixed to the cavities of the electrode plate to form spaces for receiving samples, and the samples are introduced into the wells respectively.
Such a base plate is disposable and hence can prevent contamination. The base plate may comprise only a single well, xe2x80x9cnxe2x80x9d number of wells aligned with each other, or wells two-dimensionally arranged in xe2x80x9cnxe2x80x9d number of rows and xe2x80x9cmxe2x80x9d number of columns. The material for forming the base plate is suitably prepared from universal engineering plastic such as polypropylene or polyethylene having chemical resistance and exceptional formability, in consideration of the disposability.
When employing this sample plate, voltage application to a plurality of wells becomes possible without employing a complicated electrode wiring structure. Sample injection into capillary columns of an electrophoretic apparatus employing a multi-capillary array having a number of capillary columns especially becomes simplified.
In a multi-capillary electrophoretic apparatus according to the present invention employing this sample plate, two-dimensionally arranged capillary column ends are downwardly set on a sample injection side of a mulb-capillary array migration part, the aforementioned sample plate having two-dimensionally arranged wells storing samples and a migration reservoir storing a migration buffer solution for applying a voltage to all capillary columns are arranged under the capillary column ends in correspondence to the arrangement of the capillary column ends, and the sample plate and the migration reservoir are movable for switching and bringing either one of these into contact with the capillary column ends.
In the multi-capillary electrophoretic apparatus, the samples are introduced into the wells of the sample plate fixed to the electrode plate in sample injection into the capillary columns, and the sample plate is moved by the moving mechanism for dipping ends of the respective capillary columns in the samples stored in the wells. Thereafter a voltage is applied between the electrode plate of the sample plate and other ends of the capillary columns and the samples are electrophoretically injected into the capillary columns. After sample injection, the sample plate is separated from the ends of the capillary columns by the moving mechanism, and the ends of the capillary columns are dipped into the buffer solution contained in the migration reservoir. Thereafter a voltage is applied between buffer solutions in reservoirs on both ends of the capillary columns thereby performing migration. Thus, operations from sample injection into the capillary columns to migration are automatically performed.
In this way, the multi-capillary electrophoretic apparatus employing the aforementioned sample plate according to the present invention for sample injection and comprising the moving mechanism switching and bringing either the sample plate or the migration reservoir into contact with the capillary column ends can automatically perform operations of sample injection into the capillary columns and migration.
A method according to the present invention for attaining the second objective of suppressing deterioration of migrational separability is adapted to control voltage application at the time of injecting a sample into a capillary column with voltage application to gradually raise the voltage when starting voltage application and gradually lower the voltage when finishing voltage application. As an apparatus, an applied voltage control part controlling voltage application in the aforementioned manner is provided.
Thus, stress of a gel located on a capillary column end can be reduced by suppressing abrupt heat generation and reduction. Furthermore, generation of large electroosmosis flow can be suppressed for preventing the gel from being forced out from the capillary column end. Consequently, migrational separability can be improved.
Another inventive method for attaining the second objective is a method of providing a separation carrier for preliminary separation between a sample and an end of a capillary column, injecting the sample into the capillary column through the preliminary separation carrier and removing the preliminary separation carrier before starting electrophoresis.
A capillary electrophoretic apparatus according to the present invention for carrying out the sample injection method comprises a preliminary separation part storing a separation carrier for preliminary separation and a sample so that the preliminary separation carrier is located between an end of an inserted capillary column and the sample.
In the sample injection method provided with the preliminary separation part, a voltage is applied in preliminary separation so that the sample starts moving toward the end of the capillary column. At this time, a macromolecule contained in the sample also starts moving toward the end of the capillary column and causes clogging on the preliminary separation carrier. At the same time, a target of analysis contained in the sample other than the macromolecule passes through the preliminary separation carrier and moves toward the end of the capillary column. When the target of analysis enters the capillary column, the preliminary separation carrier is removed and thereafter the target of analysis is separated. Thus, the macromolecule unreceivable in the separation carrier charged in the capillary column is removed in the stage of preliminary separation. Due to this, no macromolecule exists in separation analysis, and a separation pattern can be prevented from deterioration resulting from clogging.
The foregoing and other objects, features, aspects and advantages of the present invention will become more apparent from the following detailed description of the present invention when taken in conjunction with the accompanying drawing.